Fig. 6. XSOX3 effects on Xnr5. The 200bp Xnr5/firefly-luciferase reporter together plasmid encoding Renilla luciferase were injected into either the dorsal or ventral (A) or animal or vegetal (B) hemispheres of fertilized eggs. Embryos were homogenized at stage 9 and luciferase activities were measured. Activity was normalized using Renilla luciferase levels and the dorsal activity was set to 1 in (A), whereas vegetal activity was set to 1 in (B). (C) Coinjection of XSOX3 wild-type or m7 RNA (1 ng) activated the Xnr5 reporter to a similar extent on both dorsal and ventral sides of the embryo; m8 RNA (1 ng) had no effect on either side of the embryo. (D) Injection of XSOX3 wild-type or m7 RNA led to activation of the Xnr5 reporter in both animal and vegetal hemispheres; the m8 polypeptide produced no significant activation of the Xnr5-luciferase promoter. (E) WT Xnr5' is the sequence of the distal Lef/TcfA site of the Xnr5 promoter identified by Hilton and Old (E. Hilton and R. Old, unpublished). It contains two SOX binding sites (blue boxes marked SOXa and SOXb) and a LEF/TCF site (red box marked LEF/TCF). MUT1 removes the SOXa site, leaving the SOXb and LEF/TCF sites intact. MUT2 removes the SOXb and LEF/TCF sites, leaving the SOXa site intact. MUT3 removes both SOX sites with no effect on the LEF/TCF site. MUT4 removes both SOX sites, and would remove the LEF/TCF site were it oriented TTGTTTG rather than GTTTGAT. (F) DNA fishing of stage-8 embryonic lysates was used to analyse these mutations. After SDS-PAGE and blotting, the blots were cut. The upper XTCF3 containing region was probed with anti-XTCF3n, the lower XSOX3-containing region was probed with anti-XSOX3c. Neither polypeptide bound to streptavidin beads in the absence of biotinylated DNA (SA beads). Both XTCF3 and XSOX3 were bound to wild-type Xnr5 DNA (wt). Both XTCF3 and XSOX3 bound to the MUT1 DNA (M1), which eliminates the SOXa site. Binding of XTCF3 was eliminated by the MUT2 mutation (M2), but XSOX3 binding remained. Little or no XSOX3 bound to MUT3 (M3) or MUT4 (M4), which eliminate both SOX sites, although both bound XTCF3. XSOX3 but little XTCF3 bound to DC5, whereas the TCF sequence bound XTCF3 but little XSOX3. (G) In whole embryos, the wild-type Xnr5p reporter is activated by co-expression of XSOX3. Removal of SOXa and SOXb binding sites (MUT4) abolishes this activation, whereas removal of the TcfA and TcfB sites, either alone or together leaves the reporter responsive to XSOX3, although the TcfA mutation alone or together with the TcfB mutation reduces responsiveness to XSOX3, presumably because this mutation also removes the SOXb binding site.

Return to article